For several years our laboratory has focused on the role of orphan nuclear receptors in endocrine development and physiology with an emphasis on members of subfamily V that includes steroidogenic factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and the Drosophila Ftz-F1. Broadly speaking, these receptors participate in developmental events as well as in adult functions, including steroid and bile acid homeostasis. However, it remained unclear if subfamily V orphan nuclear receptors are ligand-dependent. We recently obtained the high-resolution crystal structure of the ligand binding domain (LBD) of LRH-1 (Sablin et al., 2003), and find that LRH-1 adopts an active conformation in the absence of ligand. Knowing this, we have pursued how ligand-independent mechanisms, such as post-translational modifications, control receptor activity. In ongoing funded work, we are exploring the structural and biological consequences of receptor phosphorylation, given that SF-1 and LRH-1 are phosphorylated in the C-terminal hinge region. Another posttranslational modification known to alter transcriptional activity is sumoylation or sumo (small ubiquitin-related modifier) conjugation of proteins, similar to ubiquitination - both SF-1 and LRH-1 are sumoylated proteins. Moreover, mutating the key acceptor lysines (K119/K194) in the hinge domain of SF- 1 increases receptor activity implying that sumoylation represses receptor-mediated transcription. These preliminary data prompt three aims: 1) What is needed to achieve full receptor sumoylation and is subnuclear localization and/or promoter occupancy affected by sumoylation? 2) What protein machinery associated with receptor sumoylation mediates repression? And lastly, 3) what are the functional consequences of loss of function sumoylation receptor mutants in Drosophila and in mice? These studies should provide mechanistic insights in SF-1 and LRH-1 activity, and as such, may provide new avenues for modulating orphan receptor activity. Additionally, given that sumoylation of regulatory factors is a new and emerging area in transcriptional regulation, our results will be of interest to the general field of gene regulation. [unreadable] [unreadable]